Four serotypes of dengue virus (DV1-4) circulate globally, causing more human illness than any other mosquito-borne virus. DV infection results in dengue fever, an acute debilitating febrile illness, or more severe life-threatening forms of th disease. DV infection confers life-long immunity against re-infection with the same serotype, but may protect against or enhance subsequent infection with a heterologous serotype. Despite the risk and the cross-protection associated with secondary DV infections, very little is known about 2nd, 3rd, or 4th DV infections specifically, largely because cohort studies with appropriate longevity have not existed. We have a unique opportunity to leverage the ICIDR influenza cohort and existing infrastructure from previous and ongoing NIH-supported studies in Managua, Nicaragua, to extend the Pediatric Dengue Cohort Study (PDCS), now arguably the longest running continuous cohort in the dengue field. The PDCS has been ongoing since August of 2004, with high-quality laboratory, epidemiological, clinical, and operational infrastructure, together with community buy-in and support from the Nicaraguan Ministry of Health. The longevity of the cohort allows specific questions about 3rd and 4th DV infections to be addressed for the first time. The resulting data have important implications for vaccine design and understanding dengue immunopathogenesis and natural protection. We will continue to follow 3,300 children 2-14 years old in Managua and examine epidemiological, clinical, and immunological characteristics of 3rd/4th vs. 2nd DV infections. This is a unique opportunity to leverage existing research infrastructure and resources to continue a long-term prospective cohort study in Nicaragua to answer critical questions about dengue that have direct implications for vaccine development. Our data from the PDCS over the past 7 years demonstrates the feasibility of identifying sufficient repeat DV infections and obtaining the samples for Aims 1-3. The PI, Director of the National Virology Laboratory (NVL) and Director of the Nicaraguan Virology Program at the Instituto de Ciencias Sostenibles in Managua, has collaborated closely with Dr. Eva Harris at the University of California (UC), Berkeley, for over 16 years. We have preliminary data supporting all the proposed experiments and methodologies and are well- poised to perform the studies outlined. Importantly, all the proposed work will be conducted in Nicaragua, with continued scientific interaction and training with the Harris Laboratory at UC Berkeley. Aim 1 will identify repeat DV infections in the cohort and determine risk factors for infection outcome in 3rd/4th vs. 2nd DV infections. Aim 2 will use longitudinal statistical methods to evaluate clinical signs and symptoms associated with 3rd/4th DV infections as compared to 1st and 2nd infections. In Aim 3, we will characterize the B cell and humoral immune response and analyze the relation of serum avidity and breadth and quality of DV-specific B cells to DV infection number and outcome. These aims address critical concepts in the dengue field that have never been carefully characterized, and the results are highly relevant for vaccine development